Expression system for large functional proteins

ABSTRACT

The present invention provides a system and method for expressing functional ABC (ATP-binding cassette) proteins, from the ABCA subfamily, in a host cell. A system comprises two or more expression vectors each comprising a nucleic acid molecule encoding one or more domains of an ABC transporter gene and a means for expressing the nucleic acid molecule. Each expression vector of the system includes a nucleic acid molecule that encodes a domain that is functionally complementary to domains contained in the other expression vectors of the system but when taken together comprise the full ABC transporter gene. Co-transfection of the expression vectors into a host cell provides co-expression of each of the domains of the protein which associate to form an ABC transporter protein having functional characteristics of the full-length protein.

FIELD OF THE INVENTION

The present invention relates to the field of protein expression. Inparticular, the present invention relates to the expression of largefunctional proteins.

BACKGROUND

ABC (ATP-binding cassette) transporters represent the largest family ofmulti-spanning membrane proteins. These proteins bind ATP and use theenergy to drive the transport of specific substrates across cellmembranes. The chemical nature of the substrates handled by ABCtransporters is extremely diverse, including drugs, lipids, peptides,metabolites and ions, yet ABC transporters are highly conserved.

Proteins are classified as ABC trasporters based on the sequence andorganization of their nucleotide-binding domain(s) (NBDs), which areresponsible for binding and hydrolyzing ATP. The NBDs are highlyconserved and contain characteristic motifs; Walker A and B, found inall ATP-binding proteins, as well as the C motif unique to the ABCtransporters. Typically, ABC transporters consist of four “core”domains, two multi-spanning membrane domains (MSDs) that serve as apathway for the translocation of a substrate across membranes and twoATP-binding cassettes or nucleotide binding domains (NBDs) that providethe energy for substrate transport (Higgins, C. F. (1992) Annu Rev CellBiol 8, 67-113). In eukaryotic ABC transporters, these domains aretypically found either on a single long polypeptide chain (fulltransporters) as in the case of CFTR and the multi-drug resistanceproteins, P-glycoprotein and MRP1, or as a complex of two identical orsimilar ‘half molecule’ subunits each having a MSD and a NBD (halftransporters), as found in the TAP1/rAP2 ABC transporter associated withpeptide antigen processing. ABCR belongs in the first category since itconsists of a single 2273 amino acid polypeptide comprised of twotandemly arranged halves (Illing, M., Molday, L. L., and Molday, R. S.(1997) J Biol Chem 272(15), 10303-10; Alliknets, R., Singh, N., Sun, H.,Shroyer, N. F., Hutchinson, A., Chidambaram, A., Gerrard, B., Baird, L.,Stauffer, D., Peiffer, A., Rattner, A., Smallwood, P., Li, Y., Anderson,K. L, Lewis, R. A., Nathans, J., Leppert, M., Dean, M., and Lupski, J.R. (1997) Nature Genet. 15, 236-246). Each half contains a MSD followedby a cytoplasmic NBD. A distinguishing feature of ABCR and other membersof the ABCA subfamily is the presence of a large exocytoplasmic(extracellular/lumen) domain that connects the first transmembranesegment to the multi-spanning membrane domain in each half of theprotein (Illing, M., Molday, L. L., and Molday, R. S. (1997) J Biol Chem272(15), 10303-10; Bungert, S., Molday, L. L., and Molday, R. S. (2001)J Biol Chem 276(26), 23539-46; Fitzgerald, M. L., Morris, A. L., Rhee,J. S., Andersson, L. P., Mendez, A. J., and Freeman, M. W. (2002) J BiolChem 277(36), 33178-87).

There are 48 or so mammalian ABC transporters that are known, and thesehave been divided into subfamilies based on similarity of gene structure(full or half transporters), order of the domains, and on sequencehomology in the NBDs and MSDs. The mammalian ABC transporters have beendivided into seven subfamilies: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, andABCG, each comprising members exhibiting a particular function (Dean, M.Rzhetsky, A., Allikmets, R. (2001) Genome Research 11:1156-1166).Mutations in the genes encoding many of these 48 or so ABC transportersare associated with a variety of inherited diseases such as cysticfibrosis, adrenoleukodystrophy, Tangier disease, and obstetriccholestasis. As well, overexpression of certain ABC transporters is themost frequent cause of resistance to cytotoxic agents includingantibiotics, antifungals, herbicides, and anticancer drugs (Higgins etal. (2001) Science. 293:1782-1784).

ABCA

The human ABCA subfamily comprises 12 full transporters. The ABCAsubfamily contains some of the largest ABC genes, several of which areover 2,100 amino acids long. Two members of this subfamily, the ABCA1and ABCA4 (ABCR) proteins, have been extensively studied. The ABCA1protein is involved in disorders of cholesterol transport and HDLbiosynthesis. The ABCA4 (ABCR) protein, also known as the rim protein,is implicated in the ATP-dependent transport of all-trans retinal acrossphotoreceptor disc membranes (Sun et al. J. Biol. Chem. (1999)274:8269-8281; Weng, J., Mata, N. L., Azarian, S. M., Tzekov, R. T.,Birch, D. G., and Travis, G. H. (1999) Cell 98, 13-23). Loss in ABCRfunction has been associated with a number of retinal degenerativediseases that cause the loss of vision (Illing, M., Molday, L. L., andMolday, R. S. (1997) J Biol Chem 272(15), 10303-10; Allikmets et al.(1997) Nature Genet. 15, 236-246). For example, over 200 differentmutations in ABCR are responsible for Stargardt macular dystrophy, anautosomal recessive retinal degenerative disease that affects over20,000 individuals in North America (Allikmets et al. (1997) NatureGenet. 15, 236-246). Mutations in the ABCR gene are also responsible fora variety of related retinal degenerative diseases including cone-roddystrophy, retinitis pigmentosa and some forms of age-related maculardegeneration, the most common form of visual impairment in the elderly.

ABCB

The ABCB subfamily comprises eleven transporter members including bothfull and half transporters. The members of the ABCB subfamily have beenimplicated in multidrug resistance (MDR). Of note is the ABCB1transporter which is characterized by its ability to confer a MDRphenotype to cancer cells.

ABCC

The ABCC subfamily contains 12 full transporter members with a diversefunctional spectrum that includes ion transport, cell-surface receptor,and toxin secretion activities. For example, the CFTR (ABCC7) protein isa chloride ion channel that plays a role in all exocrine secretions;mutations in CFTR cause cystic fibrosis.

ABCD

The ABCD subfamily contains four member proteins. All of thesetransporters are half transporters located in the peroxisome where theyfunction in the regulation of very long chain fatty acid transport.

ABCE and ABCF

The ABCE and ABCF subfamilies contain gene products that haveATP-binding domains that are clearly derived from ABC transporters butthey have no MSD and are not known to be involved in any membranetransport functions. The ABCE subfamily is solely composed of theoligo-adenylate-binding protein, a molecule that recognizesoligo-adenylate and is produced in response to infection by certainviruses. The ABCF subfamily includes three members. The bestcharacterized member, ABCF1, is associated with the ribosome and appearsto mediate the activation of the eIF-2α kinase.

ABCG

The ABCG subfamily is composed of six members, all of which are halftransporters. The mammalian ABCG1 protein is involved in cholesteroltransport regulation. Other ABCG members include ABCG2, adrug-resistance gene; ABCG5 and ABCG8, coding for transporters ofsterols in the intestine and liver.

Genes encoding most mammalian ABC transporters are very large in sizecoding for transporters that are typically between 120 kDa to 250 kDa insize. The human ABCR gene, for example, is over 6.8 kb in size and codesfor a protein of 2,272 amino acids which is expressed specifically inrod and cone photoreceptor cells of the human retina (Molday, L. L.,Rabin, A. R., and Molday, R. S. (2000) Nat Genet 25(3), 257-8). Due totheir large size, most ABC transporter genes cannot be readily packagedinto standard expression vectors for transgenic expression of thisfamily of proteins.

Most expression vector systems are limited in the size of geneticmaterial which may be inserted. For example, recombinantadeno-associated viral (rAAV) vectors, which are useful vectors for genetherapy applications, have an insert capacity of 4.9 kb, which mustinclude not only the gene, but the necessary promoters and regulatoryelements as well. This limits the types of genes that may be effectivelypackaged into expression vectors for successful transfection of hostcells. As a result, there is a need for transgenic expression systemscapable of mediating the transfer and expression of large proteins suchas the ABC transporters.

One example that circumvents the problem of delivering transgenes thatexceed the normal packaging size of the expression vector, is providedby WO 01/25465 A1. The method comprises splitting either components ofthe transcription regulatory unit or the transgene itself and packagingthese parts in two recombinant adeno-associated viral (rAAV) vectors.Co-infection with both rAAV vectors is described to result in thereconstruction of intact expression cassettes through inverted terminalrepeat mediated intermolecular concatamerization. This method islimited, however, to expanding the packaging capacity of the viralvector system at the nucleotide level.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a system and method forexpressing an ABC transporter in a host cell.

In accordance with one embodiment of the present invention, there isprovided a nucleic acid composition for expression of a functionalmember of the ABCA subfamily of ABC transporters in a host cell, saidnucleic acid composition comprising two or more different nucleic acidmolecules, each nucleic acid molecule encoding one or more domains of anABC transporter, wherein said at least one of the domains encoded byeach nucleic acid molecule are functionally complementary.

In accordance with another embodiment of the present invention, there isprovided a method of expressing a functional member of the ABCAsubfamily of ABC transporters in a host cell comprising transforming ortransfecting said host cell with the nucleic acid composition of theinstant invention.

In accordance with another embodiment of the present invention, there isprovided a system for expressing a member of the ABCA subfamily of ABCtransporters in a host cell comprising two or more expression vectors,each expression vector comprising a different nucleic acid molecule andeach nucleic acid molecule encoding one or more domains of an ABCtransporter, wherein said at least one of the domains encoded by eachnucleic acid molecule is a functionally complementary domain, andwherein, upon co-expression in said host cell, the functionallycomplementary domains associate to provide a functional ABC transporter.

In accordance with a further embodiment of the present invention, thereis provided a host cell comprising the nucleic acid composition orsystem of the instant invention.

In accordance with a further embodiment of the present invention, thereis provided a method for expressing a member of the ABCA subfamily ofABC transporters in a host cell comprising:

-   -   (a) transforming or transfecting said host cell with two or more        expression vectors, each expression vector comprising a        different nucleic acid molecule and each nucleic acid molecule        encoding one or more domains of an ABC transporter; and    -   (b) culturing said host cell under conditions that allow for        expression of said one or more domains.

In accordance with another embodiment of the present invention, there isprovided a method of treating a mammal in need of a functional ABCtransporter comprising administering to said mammal an effective amountof the nucleic acid composition or system of the instant invention.

In accordance with another embodiment of the present invention, thereare provided pharmaceutical compositions comprising the nucleic acidcompositions or system of the instant invention.

In accordance with a further embodiment of the present invention, thereis provided a kit for expressing an ABC transporter in a host cellcomprising:

-   -   (a) the nucleic acid composition or the system of the instant        invention;    -   (b) one or more containers, and optionally    -   (c) instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Immunofluorescence localization of ABCR in COS-1 cells Cellstransfected with constructs coding for the full-length ABCR, N Half(amino acids 1-1325), C Half (amino acids 1326-2273), both halves (NCHalves) were labeled with Rim5B4 (which binds an 8 amino acid epitope inNBD1 of the human ABCR; N-half) or Rim3F4 (which binds 9 amino acidsnear the C-terminus of ABCR) and Cy3 conjugated anti-mouse immunogloubinfor analysis by immunofluorescence microscopy. Full-length ABCR andco-expressed NC halves localize to both intracellular vesicles and theendoplasmic reticulum (ER)-Golgi network. The N and C halves localizepredominantly in the ER-Golgi network.

FIG. 2. The N- and C-terminal halves of ABCR associate when co-expressedin COS-1 cells. Cells transfected with the full-length ABCR, C-half,N-half, or co-transfected with the N and C halves (NC) were harvestedand the proteins were solubilized and immunopurified on aRim3F4-Sepharose matrix. ABCR and half molecules were eluted from thematrix with 3F4 peptide and analyzed by SDS-PAGE and Western blotting.The upper blot was probed for the Rim5B4 (reactive to the N-half) andthe lower blot was probed with both Rim5B4 and Rim3F4 (reactive for theC-half). Lanes 1, 4, 7, 10 13: solubilized COS-1 cell lysate; lanes 2,5, 8, 11, 14: flow-through fraction from Rim 3F4 column; lanes 3, 6, 9,12, 15: peptide eluate. CB: Commassie blue stained gel showing purifiedfull-length ABCR and co-expressed halves. The positions of thefull-length ABCR (220 kDa), N-half (140 kDa) and C-half (110 kDa) areindicated by arrows. In lanes 13-15 (N+C), cells expressing only N-halfwere mixed with cells expressing only C-half after detergentsolublization. Under these conditions, the N-half did not co-purify withthe C-half.

FIG. 3. Both halves of ABCR are required for retinal-stimulated ATPaseactivity. Purified ABCR or half molecules were expressed in COS-1 cells,purified on Rim3F4-Sepharose or Rho1D4-Sepharose (for 1D4-tagged Nhalf), and reconstituted in liposomes. The ATPase activity was measuredas a function of all-trans retinal. Filled circle, full-length ABCR;open circle, co-expressed and co-purified N and C halves; open triangle,individually expressed and purified C-half; filled triangle,individually expressed and purified 1D4-tagged N-half. The data areaverages from at least three experiments.

FIG. 4. Lysine to methionine Walker A mutations in ABCR abolish ATPhydrolysis but not ATP binding. A. ATPase activity was measured in theabsence (white bars) or presence of 50 μM all-trans retinal (grey bars).WT, wild-type; K969M in NBD1; K1978M in NBD2; MM, K969M/K1978M doublemutant; NC_(M), N-half co-expressed with C-half containing a K1978Mmutation. B. ATP photoaffinity labeling was carried out by irradiatingmembranes from COS-1 cells expressing wild-type (WT), K969M, K1978M, orK969M/K1978M double mutant (MM) with 3 μM 8-azido-[α-³²P]ATp. Thelabeled protein was isolated with Rim3F4-Sepharose and separated on SDSgels. Left panel: gel stained with Coomassie blue. Right panel:corresponding ³²P labeling obtained with a phosphorimager. All mutantproteins were labeled with 8-azido-[α-³²P]ATP, but the double mutantbound less nucleotide.

FIG. 5. Azido-ATP photoaffinity labeling of the N and C halves of ABCR.A. Membranes from transfected cells expressing either full-length ABCR(lanes 1 & 2) or co-expressing the N and C halves (lanes 3 & 4) werephotoaffinity labeled with 1.5 μM 8-azido-[α-³²P]ATP in the absence orpresence of 1 mM ATP. The expressed protein was isolated on aRim3F4-Sepharose matrix prior to analysis by SDS-PAGE and phosphorimageanalysis. Left panel: Coomassie blue stained gel. Right panel: Azido-ATPlabeling. B. Membranes from transfected cells expressing full-lengthABCR, the N-half, the C-half, or both halves (NC halves) were labeledwith 8-azido-[α-³²P] and isolated as above. Similar amounts of proteinwere loaded in each lane of the gel as judged by staining with Coomassiebrilliant blue (not shown). C. Rod outer segment membranes wereincubated with (+) or without (−) trypsin and subsequently labeled withazido-[α-³²P]ATP. ABCR and the associated N and C complex were purifiedon a Rim3F4-Sepharose matrix. Left panel: Azido-ATP labeling of thefull-length ABCR (220 kDa) and the C-half (114 kDa); N-half was notlabeled. Right panel: Western blots labeled for the full-length ABCR andC-half with the Rim3F4 antibody and the N-half with the Rim5B4 antibody.

FIG. 6. Azido-ATP binding to the N and C-halves of ABCA1. Membranes fromcells expressing the N and C halves of ABCA1 engineered to contain the3F4 epitope in the C-half and ABCR were photoaffinity labeled withazido-[α-³²P]ATP, isolated by immunoprecipitation and analyzed on an SDSgel. Coomassie blue stained gel (left panel) and azido-ATP labeling ofco-expressed N and C halves of ABCA1 (lane 1) and ABCR (lane 2) isolatedon a Rim3F4-Sepharose matrix, and ABCR (lane 3) isolated on aRim5B4-Sepharose matrix. Molecular weight markers are shown on the left.The positions of the N and C halves are indicated by arrows on theright. Both the N and C halves of ABCA1 label with 8-azido-ATP, where asonly the C-half of ABCR is intensely labeled.

FIG. 7. Nucleotide trapping by ABCR and co-expressed N and C halves. A.Effect of orthovanadate on nucleotide trapping. Membranes fromtransfected cells were incubated with 5 μM 8-azido-[α-³²P]ATP at 37° C.,washed, and tightly bound nucleotides were UV crosslinked. Lane 1,full-length ABCR; lane 2, full-length ABCR incubated with 800 μMorthovanadate; lane 3, co-expressed N and C halves; lane 4, co-expressedN- and C-halves incubated with 800 μM orthovanadate. B. Effect oftemperature and retinal on nucleotide trapping. Lane 1, full-length ABCRlabeled at 0° C.; lane 2, full-length ABCR labeled at 37° C.; lane 3,full-length ABCR labeled at 37° C. in the presence of 50 μM all-transretinal; lane 4, co-expressed N and C halves labeled at 0° C.; lane 5,co-expressed N and C halves labeled at 37° C.; lane 6, co-expressed Nand C halves labeled at 37° C. in the presence of 50 μM all-transretinal.

DETAILED DESCRIPTION OF THE INVENTION

ABC Transporters

The present invention provides a system and method for expressing an ABCtransporter in a host cell. In accordance with the present invention,two or more functionally complementary domains from one or more ABCtransporters are co-expressed in a host cell and associate in the hostcell to form a functional ABC transporter. As used interchangeablyherein, the terms “functionally complementary domain” and “complementarydomain” refer to a discrete part of a polypeptide, i.e., a domain, thatfunctionally interacts, for example by non-covalent association, with asecond, different domain to produce a fully functional protein. Thesecond domain may be part of the same polypeptide or it may be part of aseparate polypeptide. In one embodiment, the functionally complementarydomains are from an ABC transporter that is a member of the ABCAsubfamily, which includes ABCA1, ABCA2, ABCA3, ABCA4, ABCA5, ABCA6,ABCA7, ABCA8, ABCA9, ABCA10, ABCA12, and ABCA13. In another embodiment,the functionally complementary domains are from the same member of theABCA subfamily. In a further embodiment, the functionally complementarydomains are from different members of the ABCA subfamily.

Nucleic Acid Molecules

In accordance with the present invention, nucleic acid moleculesencoding at least one functionally complementary domain of an ABCtransporter are isolated. By “isolated”, it is meant a nucleic acidmolecule of genomic, cDNA, RNA, or synthetic origin or some combinationthereof, which is no longer associated with the cell in which thenucleic acid molecule is found in nature. The nucleic acid molecules ofthis invention may be isolated from cDNA or genomic libraries ordirectly from isolated eukaryotic DNA using standard techniques [see,for example, Ausubel et al, Current Protocols in Molecular Biology,Wiley & Sons, NY (1997 and updates); Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold-Spring Harbor Press, NY (2001)]. In oneembodiment of the present invention, the nucleic acid molecule encodesone or more functionally complementary domains of an ABCA transporter.In another embodiment, the nucleic acid molecule encodes at least amulti-spanning membrane domain (MSD) of an ABCA transporter. In afurther embodiment, the nucleic acid molecule encodes a multi-spanningmembrane domain (MSD) followed by a nucleotide binding domain (NBD) ofan ABCA transporter.

Nucleic acid molecules of this invention further include sequenceshaving substantial sequence similarity to a nucleic acid encoding afunctionally complementary domain of an ABC transporter. The term“substantial similarity” or “substantial sequence similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned (with appropriate nucleotide insertions or deletions)with another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50% of the nucleotidebases. In one embodiment of the invention, substantial sequencesimilarity refers to nucleotide sequence identity in at least about 60%of the nucleotide bases. In another embodiment, in at least about 70% ofthe nucleotide bases. In other embodiments, in at least about 80%, atleast about 90%, and at least about 95-98% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap.

Nucleic acid sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol.Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258(1996); Pearson, J. Mol. Biol. 276: 71-84 (1998)). Unless otherwisespecified, default parameters for a particular program or algorithm areused. For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1.

Alternatively, substantial similarity exists when a nucleic acid orfragment thereof hybridizes to another nucleic acid, to a strand ofanother nucleic acid, or to the complementary strand thereof, underselective hybridization conditions. Typically, selective hybridizationwill occur when there is at least about 55% sequence identity. In oneembodiment of the invention, selective hybridization occurs when thereis at least about 65% sequence identity. In other embodiments, there isat least about 75%, and at least about 90% sequence identity. Sequenceidentity is measured over a stretch of at least about 14 nucleotides. Inone embodiment sequence identity is measured over at least 17nucleotides. In other embodiments, over at least 20, 25, 30, 35, 40, 50,60, 70, 80, 90 and 100 nucleotides.

Nucleic acid hybridization will be affected by such conditions as saltconcentration, temperature, solvents, the base composition of thehybridizing species, length of the complementary regions, and the numberof nucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. “Stringenthybridization conditions” and “stringent wash conditions” in the contextof nucleic acid hybridization experiments depend upon a number ofdifferent physical parameters. The most important parameters includetemperature of hybridization, base composition of the nucleic acids,salt concentration and length of the nucleic acid. One having ordinaryskill in the art knows how to vary these parameters to achieve aparticular stringency of hybridization. As a general guideline,stringent washing conditions tend to fall within the ranges: 1-3×SSC,0.1-1% SDS, 50-70° C. with a change of wash solution after about 5-30minutes.

As defined herein, nucleic acid molecules that do not hybridize to eachother under stringent conditions are still substantially similar to oneanother if they encode polypeptides that are substantially identical toeach other. This occurs, for example, when a nucleic acid molecule iscreated synthetically or recombinantly using high codon degeneracy aspermitted by the redundancy of the genetic code.

It will be recognized by one of ordinary skill in the art that nucleicacids of this invention may be modified using standard techniques ofsite specific mutagenesis or PCR, or modification of the sequence may beaccomplished in producing a synthetic nucleic acid sequence. Suchmodified sequences are also considered in this invention. For example,due to the degeneracy of the genetic code, which is well-known to theart (i.e., for many amino acids, there is more than one nucleotidetriplet which serves as the codon for the amino acid) codons may bechanged such that the nucleic acid sequence encodes the same amino acidsequence, or alternatively, codons may be altered such that conservativeamino acid substitutions or substitutions of similar amino acids resultwithout affecting protein function.

The present invention also contemplates genetic engineering of thenucleic acid molecules encoding a functionally complementary domain suchthat one or more of the encoded amino acids are substantially altered.Genetic engineering techniques are standard in the art. The insertion orsubstitution of amino acids can be accomplished without adverselyaffecting the function of the domain (for example, by altering aminoacids at one or more positions remote from the functional region(s) ofthe protein), or the inserted or substituted amino acid(s) may enhancethe function of the domain, for example, inserted or substituted aminoacid(s) may enhance the ATP binding ability or ATPase activity of theassociated protein, or they may enhance the association between twodomains. Alternatively, the inserted or substituted amino acids mayconstitute a marker peptide or tag, such as an epitope.

In one embodiment of the present invention, the nucleic acid moleculeencoding a functionally complementary domain is genetically engineeredto include an epitope. In another embodiment, the nucleic acid moleculeencoding a functionally complementary domain is genetically engineeredto include a 3F4, 5B4 or 1D4 epitope.

The present invention also contemplates nucleic acid molecules encodinga functionally complementary domain fused to a heterologous nucleic acidencoding a heterologous polypeptide. Typically such heterologous nucleicacids are fused in frame to the 5′ or 3′ end of the nucleic acidencoding the functionally complementary domain and are thus capable ofexpressing a fusion protein comprising the functionally complementarydomain and the heterologous polypeptide. It will be understood that suchheterologous polypeptides will not interfere with the functioning of thefunctionally complementary domain. Examples of useful heterologouspolypeptides that may be included in the fusion proteins of the presentinvention include those designed to facilitate purification and/orvisualization of expressed functionally complementary domains.

Unless otherwise specified, the nucleic acid molecules of the presentinvention are prepared in such a manner that the intrinsic activity ofthe encoded domain is retained. The nucleic acid molecules encodingdifferent functionally complementary domains of an ABC transporter canbe used directly to transform an appropriate host cell or they may befirst incorporated into an appropriate expression vector. Methods oftransforming host cells with “naked” nucleic acid molecules are known inthe art and include, but are not limited to, direct injection of thenaked nucleic acid molecule (Felgner, P. L. and G. Rhodes, (1991) Nature349:351-352; U.S. Pat. No. 5,679,647) or the nucleic acid moleculeformulated in compositions with other agents which may facilitate itsuptake by the cell, including saponins (U.S. Pat. No. 5,739,118) andcationic polyamines (U.S. Pat. No. 5,837,533); use of microparticlebombardment (for example, by use of a “gene gun”; Biolistic, Dupont);coating or complexing the nucleic acid with lipids, cell-surfacereceptors or transfecting agents and encapsulation in liposomes,microparticles or microcapsules.

The nucleic acid molecule can be operably linked to one or moreregulatory elements that enhance expression of the encoded ABC domain.“Regulatory elements” or “regulatory sequences” refer to polynucleotidesequences that are necessary to effect the expression of coding and noncoding sequences to which they are linked, or that enhance transcriptionor translation of the sequences, stabilize the transcribed mRNA orotherwise contribute to the efficient expression of the encodedpolypeptide. The nature of such regulatory elements differs dependingupon the host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such regulatory elements includepromoters and transcription termination sequence. Regulatory elementscan further include enhancers, internal ribosomal entry sites andpolyadenylation signals. Specific initiation signals may also berequired for efficient translation of inserted nucleic acid sequences.As is known in the art, these signals include the ATG initiation codonand adjacent sequences. A minority of genes have a translationinitiation codon having the sequence 5′-GTG, 5′-TTG or 5′-CTG, and5′-ATA, 5′-ACG and 5′-CTG have been shown to function in vivo. Thesealternative initiation codons are also contemplated by the presentinvention.

One skilled in the art will appreciate that selection of suitableregulatory elements is dependent on the host cell chosen for expressionof the nucleic acid and that such regulatory elements may be derivedfrom a variety of sources, including bacterial, fungal, viral, mammalianor insect genes. The term “regulatory elements” is intended to include,at a minimum, components whose presence can influence expression of theinserted nucleic acid sequences, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

Persons of skill in the art will understand that a first nucleic acidsequence is “operably linked” with a second nucleic acid sequence whenthe first nucleic acid sequence is placed in a functional relationshipwith the second nucleic acid sequence. For instance, a promoter isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequences. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein coding regions, maintain the correct reading frame.

A promoter, as used herein, is a DNA sequence in a gene, usually (butnot necessarily) upstream (5′) to its coding sequence, which controlsthe expression of the coding sequence by providing the recognition forRNA polymerase and other factors required for proper transcription. Thetype of promoter is dependent upon the vector and the host cell selectedand can be readily determined by one skilled in the art. The promotercan be of prokaryotic and eukaryotic origin, or it may be the nativepromoter for the ABC transporter gene. In one embodiment of the presentinvention, the promoter is a eukaryotic promoter. Examples of suitableeukaryotic promoters include inducible eukaryotic promoters, e.g.tetO-minimal CMV, inducible human metallothionein IIa geneenhancer/promoter, and constitutive eukaryotic promoters e.g. CMVpromoter, SV40 late promoter, RSV LTR (rous sarcoma virus long terminalrepeat) promoter, and BGH bovine growth hormone) promoter, although manyother promoter elements well known in the art may be employed in thepractice of the invention.

Expression Vector

In accordance with one embodiment of the present invention, nucleic acidmolecules each encoding at least one functionally complementary domainof an ABC transporter are each separately incorporated into anexpression vector. Examples of suitable expression vectors include, butare not limited to, plasmids, phagemids, cosmids, bacteriophage,bacterial artificial chromosomes (BAC), yeast artificial chromosomes(YAC), baculoviruses, viral vectors (such as replication defectiveretroviruses, adenoviruses and adeno-associated viruses) or DNA viruses.In one embodiment of the present invention, the nucleic acid encodingthe ABC transporter domain is cloned into a plasmid. In anotherembodiment, the nucleic acid is cloned into a viral vector.

In one embodiment of the present invention, each vector comprises anucleic acid molecule encoding a functionally complementary domain of anABC transporter operably linked to one or more regulatory elements.“Regulatory elements” contemplated by the present invention for thispurpose include those described above and may be associated with thenucleic acid prior to insertion into the vector or they may beassociated with the vector.

Recombinant expression vectors can be constructed by standard techniquesknown to one of ordinary skill in the art and found, for example, inSambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. Avariety of strategies are available for ligating molecules of DNA, thechoice of which depends on the nature of the termini of the DNAmolecules and can be readily determined by persons skilled in the art.The vectors of the present invention may also contain other heterologousnucleic acid sequences to facilitate vector propagation and selection inhost cells. Coding sequences for selectable markers, and reporter genesare well known to persons skilled in the art.

Transformation or Transfection into a Host Cell

The recombinant expression vectors of the present invention areintroduced into a host cell capable of expressing the protein codingregion contained in each of the recombinant expression vectors. Theprecise host cell used is not critical to the instant invention and windepend upon the expression vector selected. Examples of suitable hostcells include, but are not limited to, prokaryotic host cells (e.g., E.coli or B. subtilis) and eukaryotic host cells (e.g., Saccharomyces orPichia; mammalian cells, e.g., COS, NIH 3T3, CHO, BHK, 293, or HeLacells; insect cells or plant cells). In one embodiment of the presentinvention, the host cell is of mammalian origin.

The expression vectors can be introduced into a suitable host cell viaconventional transformation or transfection techniques. The terms“transformation” and “transfection” refer to techniques for introducingforeign nucleic acid into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, microinjection and viral-mediatedtransfection. Suitable methods for transforming or transfecting hostcells can for example be found in Sambrook et al. (Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press(1989)), and other laboratory manuals.

The ABC transporter proteins of the present invention can optionally bepurified from the host cells by standard techniques known in the art. Toconfirm the presence of the preselected DNA sequence in the host cell, avariety of assays may be performed. (see, for example, Ausubel et al.,Current Protocols in Molecular Biology, Wiley & Sons, NY). Such assaysinclude, for example, “molecular biological” assays well known to thoseof skill in the art, such as Southern and Northern blotting, RT-PCR andPCR; “biochemical” assays, such as detecting the presence of apolypeptide expressed from a gene present in the vector, e.g. byimmunological means (immunoprecipitations, immunoaffinity columns,ELISAs and Western blots), by conducting activity assays or other assaysuseful to identify molecules falling within the scope of the invention.

Association of Functionally Complementary Domains to form an ABCTransporter

The co-expressed functionally complementary domains can be assayed todetermine association of the domains to form a functional ABCtransporter using standard techniques known in the art. Exemplarytesting methods are outlined herein and are not intended to limit thescope of the present invention.

In accordance with the present invention, the functionally complementarydomains are considered to have associated to form a functional ABCtransporter if at least 20% of the total recombinant protein isolatedfrom a cell is in the form of the assembled transporter protein. In oneembodiment, at least 30% of the total recombinant protein from a cell isin the form of the assembled transporter protein. In other embodiments,at least 40% and at least 50% of the total recombinant protein from acell is in the form of the assembled transporter protein.

Immunofluorescence Microscopy

Functional association of the domains of a co-expressed ABC transportermay be determined, for example, by indirect immunofluorescencemicroscopy. Like a full-length ABC transporter, co-expressedfunctionally complementary domains that have associated into an ABCtransporter should exit from the ER to intracellular vesicles,indicating that the protein complex is properly folded and assembled soas to pass through the quality control system of the ER. In contrast,domains which fail to associate, or which are individually expressed,will be misfolded and are, as a result, retained in the ER.

Immunofluorescence microscopy techniques for localizing proteins withina cell are well known in the art. Typically, cells are first treatedwith a primary antibody that recognises a specific epitope within one ormore of the functionally complementary domains. The epitope may be apart of the natural sequence or it may have been genetically engineeredinto the domain as described above. The cells are then treated with asecondary antibody that specifically binds the primary antibody and thatis conjugated to a fluorescent dye. Subsequent visualization of the dyeby fluorescence microscopy allows for the localization of the expresseddomain. Examples of useful dyes for fluorescence microscopy include, butare not limited to, rhodamine, Texas red, Cy3, Cy5 and fluorescein. Theuse of two or more primary antibodies specific to different epitopes,which are either naturally present or have been engineered into theseparate co-expressed functionally complementary domains, together withsecondary antibodies each conjugated to a fluorescent dye thatfluoresces at a different wavelength permits the localization ofmultiple domains within a cell.

Membrane Insertion

The ability of the functionally complementary domains to associate andinsert into a membrane can be analyzed in vitro. Typically the expressedprotein is solubilized using detergents and then reconstituted intomembrane vesicles using standard techniques such as those described inMolday et al. (J. Biol. Chem., (1999) 274:8269-2681); Ahn and Molday(Methods in Enzymology, (2000) 315:864-879) and the Examples.

Immunoaffinity Assays

Specific epitopes naturally present or genetically engineered into oneor more of the functionally complementary domains can also be used todetermine association of co-expressed domains into an ABC transporter byimmunoaffinity assays. A monoclonal antibody directed against a definedepitope on one domain of a co-expressed functional ABC transporter canbe coupled to a suitable matrix and contacted with, for example, cellextracts from cells co-expressing the functionally complementarydomains. Subsequent washing of the matrix is followed by elution of thebound protein with a suitable releasing agent, such as a peptide form ofthe epitope. The released protein fraction can then be analyzed bystandard techniques, such as SDS-PAGE, size exclusion chromatography,native PAGE, mass spectrometry and the like. The presence in the elutedfraction of both the domain exhibiting the targeted epitope and one ormore co-expressed domains that do not exhibit the epitope is indicativeof association of the co-expressed functionally complementary domains inthe cell. A domain which has not associated to form the functional ABCtransporter protein, and which does not exhibit the epitope, will formthe “flow-through fraction,” which is removed in the wash step(s). Suchaffinity assays are known in the art, as are methods of couplingantibodies to a suitable matrix (see for example, Coligan et al., (eds.)Current Protocols in Protein Science, and Current Protocols inImmunology, J. Wiley & Sons, New York, N.Y.). Suitable matrices include,but are not limited to, various chromatographic resin beads (including,for example, Sepharose-, agarose- and cellulose-based resins),microtitre or cell culture plates, magnetic beads, and the like.

Cross-Linking Experiments

Methods of chemically cross-linking proteins are known in the art andinclude, for example, the use of cross-linking agents such asglutaraldehyde, disuccinimidyl suberate, ethylene glycolbis(succinimidylsuccinate),bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone),dithiobis(succinimidylpropionate), M-maleimidobenzoyl succinimide esterand N-hydroxysuccinimide. A large number of other cross-linking agentsare known and are commercially available (for example, from PierceBiotechnology, Rockford, Ill.). Methods of cross-linking proteins thattake advantage of the properties of enzymes and the presence of certainresidues in the protein are also known. For example, zero-ordercross-linking takes advantage of the activity of the enzymetransglutaminase to cross-link lysine and glutamine residues in theprotein that are close together in three-dimensional space.

The ability of the functionally complementary domains to associate andform an ABC transporter can thus be determined by treating membranesisolated from cells co-expressing the domains with an appropriatecross-linking agent using standard techniques and then analyzing for thecross-linked protein by SDS-PAGE. The presence of a protein having amolecular weight corresponding to the molecular weight of the native ABCtransporter is indicative of the association of the functionallycomplementary domains.

As cross-linking of the expressed ABC transporter protein may render theprotein inactive, the activity of the protein may be assayed, usingtechniques such as those described below, prior to the cross-linkingexperiments if desired. For example, the expressed transporter could befirst photoaffinity labelled with an ATP derivative (such as8-azido-ATP) and then cross-linked. Subsequent SDS-PAGE and detection ofthe labelled ATP showing that the ATP is associated only with the highmolecular weight associated transporter protein would demonstrate thatthe functionally complementary domains associate and form an activetransporter.

Functional Protein Activity

The functional activity of the ABC transporter proteins may be evaluatedby using standard techniques well-known to workers skilled in the art.For example, the ATPase activity of the co-expressed ABC transporterdomains in the presence and absence of its natural “substrate” (i.e. amolecule that the protein normally transports) can be measured todetermine functional activity. The ATPase activity exhibited by theco-expressed domains can then be compared to that of the native proteinto determine whether the co-expressed domains have associated to form afunctional transporter protein. ATPase activity assays are well-known inthe art. For example, a method of measuring ATP hydrolysis using ATPlabelled with a detectable label and thin layer chromatography isdescribed by Ahn, J., and Molday, R. S. ((2000) Methods Enzymol 315,864-79).

Detectable labels are moieties a property or characteristic of which canbe detected directly or indirectly. One skilled in the art willappreciate that the detectable label is chosen such that it does notaffect the ability of the protein to bind ATP. Suitable detectablelabels include, but are not limited to, radioisotopes, fluorophores,chemiluminophores, colloidal particles, fluorescent microparticles andthe like. Examples of labelled ATP include, but are not limited to,trinitrophenyl (TNP)-ATP (Molecular Probes, Eugene, Oreg.) and ³²P α-ATP(NEN, Boston, Mass.). One skilled in the art will understand that theselabels may require additional components, such as triggering reagents,light, and the like to enable detection of the label. In one embodimentof the present invention, the substrates are labelled with aradioisotope. In another embodiment, the substrates are labelled withthe radioisotope ³²P.

In accordance with the present invention, the co-expressed domainsexhibit at least 40% of the ATPase activity exhibited by the native ABCtransporter. In one embodiment, the co-expressed domains exhibit atleast 50% of the ATPase activity exhibited by the native ABCtransporter.

In addition, the co-expressed domains exhibit at least 40% of thesubstrate-stimulation in ATPase activity exhibited by the native ABCtransporter. In one embodiment of the present invention, theco-expressed domains exhibit at least 50% of the substrate-stimulationin ATPase activity exhibited by the native ABC transporter. In otherembodiments, the co-expressed domains exhibit at least 60%, at least 70%and at least 80% of the substrate-stimulation in ATPase activityexhibited by the native ABC transporter.

ATP binding can also be measured using standard techniques and comparedwith the ATP binding by the native transporter. For example, it has beenshown that the native (or wild-type) ABCR transporter binds ATP only inthe C-terminal half (see Examples). ATP binding by only the C terminalhalf of the co-expressed functionally complementary domains would beindicative of wild-type functionality.

The ATP binding affinity of the co-expressed functionally complementarydomains can also be determined using techniques known in the art. Themeasured binding affinity can then be compared to that of the wild-typetransporter. In general, ATP is first labelled with a detectable label.The co-expressed functionally complementary domains or the wild-typetransporter is then mixed with various concentrations of the labelledsubstrate and the amount of bound substrate is determined. Results areanalyzed by standard methods, for example through the use of Scatchardplots, and the ATP binding affinities are compared.

Methods of assaying the ability of an ABC transporter to activelytransport its substrate across a membrane are also known in the art andcan be employed to determine whether the co-expressed functionallycomplementary domains have assembled to form an active transporter. Suchtechniques typically use a substrate labelled with a detectable label,such as those described above.

Uses of the Method of the Present Invention

The method according to the present invention can be used to express afunctional ABC transporter protein in a cell that may be defective forthe protein, or to modify the existing activity of the protein in acell. The method can find use in both research and clinical settings.

The co-expressed functionally complementary domains according to thepresent invention provide for a simple method for expressing afunctional ABC transporter in vitro and are, therefore, useful forscreening purposes, for example, for small molecule inhibitors,substrates or ligands suitable for use as therapeutics. Potentialinhibitors, substrates or ligands are screened from large libraries ofsynthetic or natural compounds. Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds and are well-known in the art Synthetic compoundlibraries are commercially available from a number of companiesincluding Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex(Princeton, N.J.), Brandon Associates (Merrinack, N.H.), and Microsource(New Milford, Conn.). A rare chemical library is available from Aldrich(Milwaukee, Wis.). Combinatorial libraries are also available and can beprepared according to standard procedures. Alternatively, libraries ofnatural compounds in the form of bacterial, fungal, plant, and animalextracts are available from, e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (North Carolina), or are readily producible. Additionally,natural and synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.

The present invention thus provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof an ABC transporter, or which bind to or act as substrates for thetransporter. The method of screening may involve high-throughputtechniques. For example, to screen for agonists or antagonists, asynthetic reaction mix, a cellular compartment, such as a membrane, cellenvelope or cell wall, or a preparation thereof, comprising an ABCtransporter formed from associated co-expressed functionallycomplementary domains is incubated in the presence of labelled substrateand a candidate molecule. The ability of the candidate molecule toagonize or antagonize the ABC transporter is reflected in increased ordecreased binding or transport of the substrate, respectively.

Gene Therapy

The present invention also contemplates expression of the functionallycomplementary domains of the ABC transporter in vivo, through the use ofgene therapy techniques.

As is known in the art, gene therapy includes both ex vivo and in vivotechniques. Thus host cells can be genetically engineered ex vivo withtwo or more nucleic acid molecules (DNA or RNA) each encoding afunctionally complementary domain, with the engineered cells then beingprovided to a patient to be treated with the ABC transporter. In suchcases, the host cells are typically autologous, so as to circumventxenogeneic or allotypic rejection, and are usually administered tocomplement defects in production or activity of the ABC transporter. Thecells are typically engineered with a vector comprising the nucleic acidmolecule of interest. Such ex vivo methods are well-known in the art.Alternatively, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, administering one or more vectorscomprising the nucleic acid molecules of interest to a patient. Thenucleic acid molecules can be directly administered to a mammal bytechniques known in the art, for example, as “naked” DNA (e.g. see U.S.Pat. No. 5,679,647), associated with transfection enhancing agents (e.g.see U.S. Pat. Nos. 5,739,118 and 5,837,533) or by the use of a “genegun.” Alternatively, the nucleic acid molecules may be firstincorporated into a suitable expression vector.

A number of vectors are known in the art to be suitable for gene therapyapplications (see, for example, Viral Vectors: Basic Science and GeneTherapy, Eaton Publishing Co. (2000)). The vectors are typicallyviral-based vectors and include, but are not limited to, those derivedfrom replication deficient retrovirus, lentivirus, adenovirus andadeno-associated virus. Retrovirus vectors and adeno-associated virusvectors are currently the recombinant gene delivery system of choice forthe transfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population.Retroviruses, from which the retroviral vectors hereinabove mentionedmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus,human immunodeficiency virus, adenovirus, Myeloproliferative SarcomaVirus, and mammary tumour virus. Specific retroviruses include pLJ,pZIP, pWE and pEM, which are well known to those skilled in the art.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter, theE1A promoter, the major late promoter (MLP) and associated leadersequences or the E3 promoter, the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTR; the histone, pol III, and β-actin promoters;B19 parvovirus promoter; the SV40 promoter; and human growth hormonepromoters. The promoter also may be the native promoter for the gene ofinterest. The selection of a suitable promoter will be dependent on thevector, the host cell and the encoded protein and is considered to bewithin the ordinary skills of a worker in the art.

The development of specialized cell lines (termed “packaging cells”)which produce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterised for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid molecule ofthe invention and renders the retrovirus replication defective. Thereplication defective retrovirus is then packaged into virions that canbe used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable packaging viruslines for preparing both ecotropic and amphotropic retroviral systemsinclude Crip, Cre, 2 and Am. Other examples of packaging cells include,but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X,VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines asdescribed in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990).

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (for example, lactose toconvert the env protein to an asialoglycoprotein), as well as bygenerating fusion proteins ((for example, single-chain antibody/envfusion proteins). This technique, while useful to limit or otherwisedirect the infection to certain tissue types, can also be used toconvert an ecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the nucleic acid molecules of the inventioncontained in the vector.

Another viral vector useful in gene therapy techniques is anadenovirus-derived vector. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 d1324 or other strains ofadenovirus (for example, Ad2, Ad3, Ad7 etc.) are well known to thoseskilled in the art. Recombinant adenoviruses can be advantageous incertain circumstances in that they can be used to infect a wide varietyof cell types, including peripheral nerve cells. Furthermore, the virusparticle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (for example,retroviral DNA). Moreover, the carrying capacity of the adenoviralgenome for foreign DNA is large (up to 8 kilobases) relative to othergene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham(1986) J. Virol. 57:267). Most replication-defective adenoviral vectorscurrently in use and contemplated by the present invention are deletedfor all or parts of the viral E2 and E3 genes but retain as much as 80%of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell16:683; Berkner et al., supra; and Graham et al. in Methods in MolecularBiology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127).

Compositions

The present invention also relates to pharmaceutical compositionscomprising the nucleic acid molecules or expression vectors comprisingthe nucleic acid molecules discussed above. Thus, the nucleic acidmolecules or expression vectors comprising the nucleic acid molecules ofthe instant invention may be employed in combination with a non-sterileor sterile carrier or carriers for use with cells, tissues, ororganisms, such as a pharmaceutical carrier suitable for administrationto a subject. Such compositions comprise, for instance, a media additiveor a therapeutically effective amount of the nucleic acid molecules orexpression vectors comprising the nucleic acid molecules of theinvention and a pharmaceutically acceptable carrier or excipient. Suchcarriers may include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

Pharmaceutical compositions and methods of preparing pharmaceuticalcompositions are known in the art and are described, for example, in“Remington: The Science and Practice of Pharmacy” (formerly “RemingtonsPharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins,Philidelphia, Pa. (2000).

Administration

The nucleic acid molecules or expression vectors comprising the nucleicacid molecules of the present invention may be employed alone or inconjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Kits

The present invention additionally provides for kits containing thenucleic acid molecules encoding the functionally complementary domainsor expression vectors comprising the nucleic acid molecules. Individualcomponents of the kit would be packaged in separate containers and,associated with such containers, can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

For therapeutic applications, the nucleic acid molecules or expressionvectors comprising the nucleic acid molecules can be in the form ofpharmaceutically acceptable compositions. When the components of the kitare provided in one or more liquid solutions, the liquid solution can bean aqueous solution, for example a sterile aqueous solution. For in vivouse, the expression construct may be formulated into a pharmaceuticallyacceptable syringeable composition. In this case the container means mayitself be an inhalant, syringe, pipette, eye dropper, or other such likeapparatus, from which the formulation may be applied to an infected areaof the animal, injected into an animal, or applied to and mixed with theother components of the kit.

The components of the kit may also be provided in dried or lyophilizedforms. When reagents or components are provided as a dried form,reconstitution generally is by the addition of a suitable solvent.

The invention now being generally described, it will be more readilyunderstood by references to the following examples, which are includedfor purposes of illustration only and are not intended to limit theinvention unless so stated.

EXAMPLES Example 1 Construction of Plasmids

cDNA fragments individually encoding the N-terminal and C-terminalhalves of the human ABCR and ABCA1 protein were generated. Each half ofthe respective genes contains a multi-spanning membrane domain followedby a nucleotide binding domain. Each cDNA fragment was subcloned intoseparate pcDNA3 expression vectors (Invitrogen).

The human ABCR cDNA was generously provided by J. Nathans, Johns HopkinsUniversity and the human ABC1 (ABCA1) was a gift of Active PassPharmaceuticals, Vancouver, B.C. The cDNAs were subcloned into themammalian expression vector pcDNA3 (Invitrogen) to produce pcABCR andpcABCA1.

ABCR

The cDNAs coding for the N-half (amino acids 1-1325) and C-half (aminoacids 1326-2273) of ABCR were constructed by PCR using the followingprimer pairs: N-half - 5′GAGCCCTGTGGCCGGCCAGCTGT [SEQ ID NO:1]G-3′ (FseI) and 5′-GCTCTAGATTACGGCGCCCCTGGGGAGCAGACAT [SEQ ID NO:2]TGG-3′ (XbaI); N-half-1D4 - 5′-GAGCCCTGTGGCCGGCCAGCT [SEQ ID NO:3]GTG-3′ (FseI) and 5′-GCTCTAGATTAGGCAGGCGCCACTTGGCTGGTCT [SEQ ID NO:4]CTGTCGGCGCCCCTGGGGAGCAGACATT GG-3′ (XbaI); C-half -5′-TGCTCCAAGCTTAGCATGGCTGCTC [SEQ ID NO:5] ACCCAGAGGG-3′ (HindIII) and5′-CAGGGGTACTCCGGAAGC-3′ (BspE1). [SEQ ID NO:6]

The restriction sites used to insert the PCR products are underlinedwith the enzyme indicated in parentheses. The bases coding for the 1D4epitope are shown in italics. The PCR products were digested with theindicated restriction enzymes and ligated into pcABCR that had beendigested with the same enzymes.

K969M and K1978M mutations were inserted by QuikChange site directedmutagenesis (Stratagene) using PfuTurbo DNA polymerase and the followingmutagenic primers (introduced mutations in bold): K969M -5′-CCACAATGGAGCTGGGATGACCAC [SEQ ID NO:7] CACCTTGTCC-3′ andGGACAAGGTGGTGGTCATCCCAGCTCCATTGTGG-3′ [SEQ ID NO:8] (JA13/JA14);K1978M - 5′-GAATGGTGCCGGCATGACAACCAC [SEQ ID NO:9] ATTCAAGATGC-3′ and5′-GCATCTTGAATGTGGTTGTCATGCCGGCACCAT [SEQ ID NO:10] TC-3′ (JA15/JA16).

The AflII-ClaI (1.9 kb) and the Eco72I (0.26 kb) fragments of theresulting PCR products containing the K969M and K1978M mutations,respectively, were cloned into the original pcABCR. For the K969M/K1978Mdouble mutant, the AflII-FseI restriction fragment of pcABCR[K1978M] wasreplaced with that of pcABCR[K969M].

ABCA1

The cDNA for the N-half-1D4 (amino acids 1-1302) of ABCA1 was made byreplacing the 4.2 kb PmlI-XbaI fragment of pcABCA1 with a 0.9 kb PCRproduct amplified with the following primers: 5′-CACATCTGGTTCTATGCC-3′[SEQ ID NO:11] and 5′-CCTCTAGATTAGGCAGGCGCCACTTGGCTGGTC [SEQ ID NO:12]TCTGTGGATTCTGGGTCTATGTC-3′ (JA24/JA25).

The cDNA coding for the C-half of ABCA1 (amino acids 1303-2261)containing the 3F4 epitope was synthesized by PCR (2.9 kb) with thefollowing primers: 5′-ACTGATGCGGCCGCGGGAACATGGAATCCAGAG [SEQ ID NO:13]AGACAGACTTG-3′ and 5′-TCCGCTAGCGTTTAAACTCATCCAGTTCGAGGG [SEQ ID NO:14]TGCAAAGGCAGATCGTATACA TAGCTTTCTTTCAC-3′ (JA29/JA30)and cloned into pCEP4 at the NotI/PmeI sites.

All PCR amplified sequences were confirmed by automated DNA sequencing.

Example 2 Transfection of COS-1 and EBNA293 Cells

The monkey kidney fibroblast cell line COS-1 was maintained in DMEM(high glucose) supplemented with 10% fetal bovine serum. Human embryonickidney EBNA293 cells (Invitrogen) were grown in the above DMEMcontaining 0.25 g/L G418. Cells were plated on 10 cm dishes andtransfected the following day with 30 μg of plasmid per dish using thecalcium phosphate method. The next day, cells were rinsed with 1 mM EDTAin PBS, pH 7.4, and supplied with complete medium for 24 h.

Example 3 Purification and Reconstitution

Membranes (from two 10 cm dishes) were solubilized in 0.5 ml of 1%Triton-X100 in Buffer A (140 mM NaCl, 20 mM Tris-HCl, pH 7.4) for 20 minon ice. For ATPase assays, the membrane preparation step was omitted andthe cell suspension was solubilized directly in Buffer B (10 mg/mlsoybean phospholipids, 10% glycerol, 1 mM dithiothreitol, 100 mM NaCl, 3mM MgCl₂, 50 mM NaHEPES, pH 7.4) containing 18 mM CHAPS. The supernatantafter a 10 min centrifugation at 40,000 rpm (TLA100.4 rotor) was mixedwith 50 μl Rim3F4 Sepharose 2B for 1 h at 4° C. The beads were washed 6times in Buffer A containing 0.2% Triton X-100 or Buffer B containing 10mM CHAPS and eluted with 4% SDS (for electrophoresis) or 0.2 mg/mlRim3F4 peptide (for reconstitution and determination of ATPaseactivity). Purified protein (24 μl) was incubated with 6 μl of 50 mg/mllipid (1:1 mixture of dioleoylphosphatidylethanolamine and brain polarlipid, by weight) and 3 μl n-octylglucoside for 30 min on ice. Themixture was diluted rapidly with 200 μl of Buffer C (1 mMdithiothreitol, 140 mM NaCl, 25 mM NaHEPES, pH 7.4) and passed through a200 μl Extracti-gel column (Pierce). The flow-through containing thereconstituted protein was used for determination of ATPase activity.

Example 4 Localization of ABCR in Transfected COS-1 Cells by IndirectImmunofluorescence Microscopy

The subcellular distribution of full length ABCR and the N and Chalf-molecules expressed in COS-1 cells was determined byimmunofluorescence microscopy (FIG. 1). Rather than localizingpredominantly in the endoplasmic reticululm (ER) and Golgi, which isexpected for transiently overexpressed intracellular membrane proteins,ABCR was typically associated with intracellular vesicles of varyingsizes. Clusters of 2-4 large vesicles were observed in some cells, whilenumerous small vesicles spread throughout the cytoplasm were seen inother cells. These intensely labeled vesicles do not appear to beartifacts since mutating a single amino acid in ABCR (D846H) changed thedistribution from vesicular to perinuclear reticular distributioncharacteristic of misfolded proteins retained in the ER. ABCR did notco-localize with a number of organelle markers (catalase forperoxisomes, LAMP-2 for late endosomes, LysoTracker for lysosomes). Theexpression pattern of the N-half or C-half when expressed alone wasmostly perinuclear indicative of ER localization. However, when the twohalves were co-expressed, a significant fraction of the protein wasfound in vesicular structures like those seen in cells transfected withwild-type, full-length ABCR.

Example 5 Functional Protein Activity

1) Association of the Two Halves of ABCR when Co-Expressed

Non-reduced samples were prepared by solubilizing cells in the presenceof 100 mM n-ethylnaleimide to prevent formation of secondary disulfidebonds. Proteins were separated on 6% polyacrylamide gels, stained withCoomassie brilliant blue, destained in 10% acetic acid and soaked inwater. The gel was dried under vacuum and exposed to a storage phosphorscreen or autoradiography film. For Western blot analysis, theelectrophoresed proteins were transferred to an Immobilon-P membranewhich was subsequently blocked in 1% nonfat milk and incubated withprimary and peroxidase-conjugated secondary antibodies. Duplicatesamples were loaded on the same gel and analyzed on Western blots usingRim3F4 (1:10 dilution) and Rim5B4 (1:100 dilution) antibodies.

The N and C halves of ABCR, each containing a transmembrane domainfollowed by an NBD, were expressed individually by single transfectionsor together by co-transfection in COS-1 cells. FIG. 2 shows Westernblots of COS-1 cell extracts, flow-through (unbound) fractions, andpeptide-eluted (bound) fractions of the expressed full-length ABCR (−220kDa) and the N (−140 kDa) and C (110 kDa) half-molecules isolated on aRim3F4-Sepharose matrix specific for an epitope near the C-terminus ofABCR(2). When the two halves were co-expressed (NC), about 50% of theN-half (detected with the Rim5B4 MAb) co-purified with the C-half(detected with the Rim3F4 MAb), while the remainder was in theflow-through fraction The N-half by itself did not bind to theRim3F4-Sepharose matrix nor did it co-purify with the C-half when the Nand C halves were individually expressed and mixed together prior toimmunoaffinity purification. Coomassie blue stained gels showed thatfull-length ABCR and co-expressed/co-purified N and C halves were thepredominant proteins observed in the peptide-eluted fraction from theRim 3F4 immunoaffinity column.

2) ATPase Activity

ATPase activity was measured as described previously (Ahn, J., Wong, J.T., and Molday, R. S. (2000) J Biol Chem 275(27), 20399-405) using 50 μM[α³²P]ATP and thin layer chromatography. The all-trans retinalconcentration was determined spectrophotometrically (λ_(383 nm)=42.88mM⁻¹ cm⁻¹). Protein concentration was estimated from the eluate beforereconstitution by laser densitometry of Coomassie blue stained gelsusing bovine serum albumin as a standard. This method gives anoverestimation of the actual protein content after reconstitution (hencelower specific activity) since recovery from the Extracti-gel column isless than 100 percent. Direct protein measurements after reconstitutionby densitometry of Western blots was about half of that in the eluate.However, the latter method gave variable results, so proteinconcentration after reconstitution was extrapolated from that in theeluate assuming 100% recovery.

ATPase Activity of Expressed N and C Halves—The basal and retinalactivated ATPase activity of full-length ABCR and the N and C halvesindividually or co-expressed in COS-1 cells was determined afterimmunoaffinity purification and reconstitution into lipid vesicles. FIG.3 shows that both the full-length ABCR and co-expressed N and C halvespurified on a Rim 3F4 column were stimulated 1-2 fold by all-transretinal. The specific activity of the full-length protein, however, wasgenerally higher than the co-expressed half molecules. In contrast, theATPase activity of the individually expressed C half and N halfcontaining a nine amino acid 1D4 epitope tag (N*) required forimmunoaffinity purification was not stimulated by retinal. The 1D4 tagdid not affect the functional interaction of the N and C halves, sinceco-expression of the N* and C halves gave similar basal and retinalstimulated activity as co-expression of the untagged N and C halves(data not shown).

3) Preferential Azido-ATP Labelling

Preparation of Membranes—Membranes from transfected cells were preparedas described previously (Bungert, S., Molday, L. L., and Molday, R. S.(2001) J Biol Chem 276(26), 23539-46). In some experiments, the protocolwas scaled down and the cell homogenate from one or two 10-cm dishes,after passing through a 26-gauge needle (6 times), was centrifuged on adiscontinuous gradient consisting of 5% and 60% sucrose for 30 min at30,000 rpm (60,000×g) in a TLS55 rotor (Beckman Optima TLultracentrifuge).

8-Azido-ATP Photoaffinity Labeling—Membranes (50-150 μg protein) in 50μl of labeling buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM MgCl₂)were incubated with 1-4 μM 8-azido-[α-³²P]ATP (Perkin-Elmer LifeScience; 20 Ci/mmol) on ice, with gentle shaking, under UV light (254μm) for 10 min at a distance of 10 cm. Ice-cold 20 mM Tris-HCl, pH 7.4,was added and the membranes were collected by centrifugation (TLA45rotor, 55000 g, 15 min). The membrane suspension (200 μl in TBS[Tris-HCl, pH 7.4, 140 mM NaCl]) was added to 200 μl of 2% Triton X-100in TBS, pH 7.4). After 30 min on ice, the cleared extract was mixed with25 μl antibody coupled to Sepharose 2B for 1-12 h at 4° C. The beadswere washed 4 times in TBS containing 0.2% Triton X-100 and elutedtwice, 30 μl each, in 4% SDS, 0.2% Triton X-100, TBS.

Trypsin Cleavage of Bovine ABCR—Bovine ROS were isolated as previouslydescribed (Molday, R. S., and Molday, L. L. (1987) J Cell Biol 105(6 Pt1), 2589-601) and treated with 1.6-4.0 μg/ml trypsin for 30 min at 0° C.(Bungert, S., Molday, L. L., and Molday, R. S. (2001) J Biol Chem276(26), 23539-46). The reaction was stopped by the addition of 5-foldexcess of soybean trypsin inhibitor.

8-Azido-ADP Photoaffiniy Labeling—Thoroughly washed ROS membranes werelabeled with 5 μM 8-azido[α-³²P]ADP (Affinity Labeling Technologies,16.8 Ci/mmol) for 15 to 30 min as described for 8-azido-ATP binding. DTrat a final concentration of 10 mM was added to quench the reaction.After 15 min, ice-cold Tris-EDTA buffer (0.5 mM EDTA, 10 mM Tris-HCl, pH7.4) was added and the membranes were washed 5 times by centrifugationat 30,000 rpm for 15 min (TLA45 rotor in a Beckman Optima TL-100centrifuge). The membranes were suspended in 50 μl of Tris-EDTA bufferand an equal volume of buffer containing trypsin (4 μg) was added. After30 min on ice, the membranes were treated with trypsin inhibitor (50μg). Cold Tris-EDTA, pH 7, buffer was added and the membranes pelletedby centrifugation (30000 rpm, 15 min in a TLA 45 rotor). The membraneswere resuspended in 50 μl of Tris-EDTA buffer, and an equal volume ofSDS sample buffer with β-mercaptoethanol was added. Thirty μl sampleswere loaded in triplicate onto three 8% SDS gels: two gels were used forWestern blot analysis with Rim3F4 and Rim5B4 antibodies, respectively,and the third gel was stained, destained, dried and analyzed for ³²Plabeling with a PhosphorImager.

To remove tightly bound nucleotides, membranes were treated as follows(Hyndman, D. J., Milgrom, Y. M., Bramhall, E. A., and Cross, R. L.(1994) J Biol Chem 269(46), 28871-7). The washed membrane pellet (4 mg)was resuspended in 1 ml of 100 mM Na₂SO₄, 50% glycerol, 3 mM MgCl₂, 50mM NaHEPES, pH 7.5, and dialyzed against 3 changes of the same buffer at4° C. (2×500 ml for 3 h each, 1000 ml overnight). The sample (100 μl)was diluted with labeling buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5mM MgCl₂). The washed pellet was resuspended and photolabeled asdescribed above.

The washed membranes (4 mg) were resuspended in 1 ml of 50 mM NaHEPES,pH 7.5, 100 mM Na₂SO₄, 50% glycerol, 3 mM MgCl₂ and 10 mM CHAPS, stirredin a glass tube at 4° C. for 1 h, and dialysed against 3 changes ofresuspension buffer. The solubilized membranes, 100 μl, were centrifugedthrough a column of Sephadex G-50 (previously equilibrated with labelingbuffer). The volume was made up to 100 μl with labeling buffer and thesample was photoaffinity labeled as described above. This was thenpassed through another column centrifugation procedure to remove unboundlabel. Half of the labeled sample was subjected to trypsin digestion asdescribed above.

To determine whether ATP binds to one or both NBDs of ABCR, membranesfrom transfected cells were photoaffinity labeled with 8-azido-[³²P]ATP,and expressed ABCR proteins were purified by immunoaffinitychromatography. FIG. 5 shows that when the N and C halves were expressedtogether, only the C-half was labeled with 8-azido-ATP. This labelingwas essentially abolished by the addition of 1 mM ATP prior tophotoaffinity labeling (FIG. 5A, lanes 2 and 4). When expressedindividually, the N-half bound ATP weakly and the C-half did not bindATP at all (FIG. 5B).

To determine if the C-half of native ABCR also selectively bound ATP,ABCR in ROS disk membranes was cleaved with trypsin to generate N and Chalf molecules of similar size (Illing, M., Molday, L. L., and Molday,R. S. (1997) J Biol Chem 272(15), 10303-10; Bungert, S., Molday, L. L.,and Molday, R. S. (2001) J Biol Chem 276(26), 23539-46) forphotoaffinity labeling with 8-azido-[³²P]ATP. Only the C-half (˜114 kDa)was labeled (FIG. 5C) as found for the co-expressed N and C halfmolecules of ABCR. Identical results were obtained when full-lengthbovine ABCR was photoaffinity labeled with 8-azido [³²P]ATP prior totrypsin cleavage (data not shown). This confirms that both theco-expressed N and C half molecules of ABCR and the native protein arefunctioning in the same way.

The possibility that more C-half than N-half is recovered on Rim3F4beads and therefore displays more azido-ATP label was investigated. Thisis unlikely to be a problem since it has already been shown that anyexcess C-half which is not associated with N-half does not label withazido-ATP (FIG. 5B). Nevertheless, FIG. 6 lane 3 shows that when the twohalves of ABCR were co-immunoprecipitated with Rim5B4 (which binds theN-half of ABCR and should only purify C-half that is bound to theN-half), the C-half is still labeled more strongly.

4) Amido-ATP/ADP Trapping by Co-expressed N and C Halves of ABCR

8-Azido-ATP Trapping—Membranes were incubated with 5 μM8-azido-[α-³²P]ATP in 50 μl of labeling buffer with or without 800 μMsodium orthovanadate for 10 min at 37° C. All-trans retinal and 50 μMDTT were added where indicated. Binding was stopped by the addition ofice-cold 20 mM Tris-HCl, pH 7.4, the membranes were collected bycentrifugation and washed one more time. Samples exposed toorthovanadate were washed in the presence of 800 μM orthovanadate. Insome experiments MGATP (10 mM) was included in the wash step, but had noeffect. The membranes were suspended in 30 μl of 20 mM Tris-HCL pH 7.4,irradiated with UV light for 10 min on ice, diluted to 200 μl with TBS,pH 7.4, and solubilized as described above for azido-ATP binding.

To gain more insight into the properties of the NBD of ABCR, trappingexperiments were carried out using 8-azido-[α³²P]ATP. As shown in FIG.7, more 8-azido ATP/ADP was trapped by ABCR under hydrolyzing conditions(37° C.) than at 0° C. This binding was not dependent on sodiumorthovanadate or influenced by the presence of all-trans retinal (FIG.7). Co-expression of the N and C halves further revealed that as withATP binding, ATP/ADP trapping occurred in NBD2 of the C-half, confirmingthat the co-expressed protein retained the functionality of the nativeABCR protein.

Example 6 8-Azido-ATP Binding by Co-expressed N and C Halves of ABCA1

It has been established that the N-terminal NBD (NBD1) of MRP1, CFTR,SUR1 or the NBD of TAP1 is responsible for high affinity ATP binding andthe C-terminal NBD (NBD2) or TAP2 is more important for ATP hydrolysisand ADP trapping. The unexpected finding that ATP binding occurs only onthe C half of ABCR, prompted the examination of the ATP bindingproperties of ABCA1, a member of the ABCA subfamily which is mostsimilar to ABCR.

Membranes from cells expressing the N and C halves of ABCA1 engineeredto contain the 3F4 epitope in the C-half and ABCR were photoaffinitylabeled with azido-[α-³²P]ATP, isolated by immunoprecipitation usingRim3F4-Sepharose 2B beads and analyzed on an SDS gel.

FIG. 6 shows that the two halves of ABCA1 were labeled equally well incontrast to ABCR. FIG. 6 also demonstrates that isolation of theco-expressed halves of ABAC1 using Rim3F4, which only binds theC-terminal half of the protein, also isolated the N half of the proteinindicating that the two halves of ABCA1 associate when co-expressed.

Example 7 Effect of Walker A Lysine-to-Methionine Mutations on ATPHydrolysis and Binding

The conserved lysine residue in the NBD Walker A motif of ABC proteinsis critical for the hydrolysis of ATP. Mutation of this lysine tomethionine in P-glycoprotein abolishes basal and drug-stimulated ATPaseactivity (35,36). With ABCR, the lysine-to-methionine substitution inthe NBD1 (K939M) and NBD2 (K1978M) or in both (K939M/K1978M)significantly reduced the basal ATPase activity of ABCR, and abolishedretinal activation (FIG. 4A). In an analogous manner, retinal-stimulatedATPase activity was also abolished when the N-half was co-expressed withthe K1978M C-half mutant (amino acid numbers represents that of thefull-length ABCR), again showing that the functionality of theco-expressed transporter and the native protein are the same.

The ATP binding of these mutants was also examined using thephotoreactive ATP analogue 8-azido-[α-³²P]ATP. The photoaffinitylabeling intensities of the single mutants (K969M and K1978M) weresimilar to wild-type ABCR relative to the amount of purified ABCRstained with Coomassie blue (FIG. 4B). A small reduction in labeling,however, was observed for the K969M/K1978M double mutant.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. A nucleic acid composition for expression of a functional member ofthe ABCA subfamily of ABC transporters in a host cell, said nucleic acidcomposition comprising two or more different nucleic acid molecules,each nucleic acid molecule encoding one or more domains of an ABCtransporter, wherein said at least one of the domains encoded by eachnucleic acid molecule are functionally complementary.
 2. The nucleicacid composition according to claim 1, wherein said two or more nucleicacid molecules are associated with a lipid.
 3. The nucleic acidcomposition according to claim 1, wherein said two or more nucleic acidmolecules are provided in separate expression vectors.
 4. The nucleicacid composition according to claim 1, wherein said two or more nucleicacids are operatively associated with one or more regulatory elements.5. A host cell comprising the nucleic acid composition according toclaim
 1. 6. A method of expressing a functional member of the ABCAsubfamily of ABC transporters in a host cell comprising transforming ortransfecting said host cell with the nucleic acid composition accordingto claim
 1. 7. A system for expressing a member of the ABCA subfamily ofABC transporters in a host cell comprising two or more expressionvectors, each expression vector comprising a different nucleic acidmolecule and each nucleic acid molecule encoding one or more domains ofan ABC transporter, wherein said at least one of the domains encoded byeach nucleic acid molecule is a functionally complementary domain, andwherein, upon co-expression in said host cell, the functionallycomplementary domains associate to provide a functional ABC transporter.8. The system according to claim 7, wherein said two or more expressionvectors further comprise one or more regulatory elements operativelyassociated with said nucleic acid molecule.
 9. The system according toclaim 7, wherein said two or more expression vectors are plasmids. 10.The system according to claim 7, wherein said two or more expressionvectors are viral vectors.
 11. The system according to claim 7, whereinsaid one or more domains comprise a nucleotide binding domain (NBD). 12.The system according to claim 11 wherein said one or more domainsfurther comprise a multi-spanning membrane domain (MSD).
 13. The systemaccording to claim 7, wherein said one or more domains comprise amulti-spanning membrane domain (MSD).
 14. The system according to claim7, wherein at least one of said domains comprise an epitope.
 15. Thesystem of claim 14, wherein said epitope is selected from the groupconsisting of: 3F4, 5B4, and 1D4.
 16. A host cell comprising the systemaccording to claim
 7. 17. A method for expressing a member of the ABCAsubfamily of ABC transporters in a host cell comprising: (a)transforming or transfecting said host cell with two or more expressionvectors, each expression vector comprising a different nucleic acidmolecule and each nucleic acid molecule encoding one or more domains ofan ABC transporter; and (b) culturing said host cell under conditionsthat allow for expression of said one or more domains.
 18. The methodaccording to claim 17, wherein said host cell is a prokaryotic cell. 19.The method according to claim 17, wherein said host cell is a eukaryoticcell.
 20. The method according to claim 17, wherein said functional ABCtransporter forms at least 20% of the total recombinant protein producedby said cell.
 21. The method according to claim 17, wherein saidfunctional ABC transporter exhibits at least 50% of the ATPase activityof the native ABC transporter.
 22. A method of treating a mammal in needof a functional member of the ABCA subfamily of ABC transporterscomprising administering to said mammal an effective amount of thenucleic acid composition according to claim
 1. 23. A method of treatinga mammal in need of a functional member of the ABCA subfamily of ABCtransporters comprising administering to said mammal an effective amountof the system according to claim
 7. 24. A pharmaceutical compositioncomprising the nucleic acid composition according to claim
 1. 25. A kitfor expressing a member of the ABCA subfamily of ABC transporters in ahost cell comprising: (a) the nucleic acid composition according toclaim 1; (b) one or more containers, and optionally (c) instructions foruse.
 26. A pharmaceutical composition comprising the system according toclaim
 7. 27. A kit for expressing a member of the ABCA subfamily of ABCtransporters in a host cell comprising: (a) the system according toclaim 7; (b) one or more containers, and optionally (c) instructions foruse.